Progenitor cells of mesodermal lineage

ABSTRACT

The invention relates to progenitor cells of mesodermal lineage and their use in therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/130,840 filed Sep. 25, 2014, which claims priority to InternationalApplication No. PCT/GB2012/051600 filed on Jul. 6, 2012, which claimspriority to Great Britain Patent Application Nos. 1111500.3; 1111503.7;1111505.2; and 1111509.4 previously filed on Jul. 6, 2011, all of whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to progenitor cells of mesodermal lineage andtheir use in therapy.

BACKGROUND TO THE INVENTION

Mesenchymal stem cells (MSCs) are multipotent, adult stem cells. MSCsdifferentiate to form the different specialised cells found in theskeletal tissues. For example, they can differentiate into cartilagecells (chondrocytes), bone cells (osteoblasts) and fat cells(adipocytes).

MSCs are used in a variety of therapies, such as the treatment ofAge-related Macular Degeneration (AMD) and myocardial infarct. Onceadministered to the patient, the MSCs typically migrate (or home) to thedamaged tissue and exert their therapeutic effects through paracrinesignaling and by promoting survival, repair and regeneration of theneighbouring cells in the damaged tissue.

Current therapies typically involve the infusion of a mixture of MSCsubtypes some of which do not migrate efficiently to the tissue ofinterest. This necessitates the use of a high cell-dose which can leadto off-target side effects and volume-related side effects. MSCs aretypically obtained from bone marrow and so it is difficult to obtainlarge amounts.

SUMMARY OF THE INVENTION

The inventors have surprisingly identified a new class of progenitorcells of mesodermal lineage (PMLs) having a specific marker expressionpattern. Homogenous populations of the PMLs of the invention can beisolated from mononuclear cells (MCs), such as peripheral blood MCs. ThePMLs are capable of efficiently migrating to and repairing damagedtissues.

The invention provides a progenitor cell of mesodermal lineage, whereinthe cell (a) expresses detectable levels of CD29, CD44, CD73, CD90,CD105 and CD271 and (b) does not express detectable levels of CD14, CD34and CD45.

The invention also provides:

-   -   a population comprising two or more progenitor cells of the        invention;    -   a pharmaceutical composition comprising (a) a progenitor cell of        the invention or a population of the invention and (b) a        pharmaceutically acceptable carrier or diluent;    -   a method of producing a population the invention, comprising (a)        culturing mononuclear cells (MCs) under conditions which induce        the MCs to differentiate into progenitor cells of mesodermal        lineage and (b) harvesting and culturing those progenitor cells        which have an expression pattern of the invention and thereby        producing a population of the invention;    -   a method of repairing a damaged tissue in a patient, comprising        administering to the patient a population of the invention,        wherein the population comprises a therapeutically effective        number of cells, and thereby repairing the damaged tissue in the        patient; and    -   a population of the invention for use in repairing a damaged        tissue in a patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an RT-PCR gel confirming the presence of CD44 and theabsence of CD34 in the PMLs of the invention.

FIG. 2 shows the results of FACS analysis on the PMLs of the invention.This confirms that the cells are positive for at least CD73 and CD90 andnegative for CD14, CD34 and CD45.

FIG. 3 shows further results from FACS analysis, namely the histogramsfor CD90 (top) and CD73 (bottom).

FIGS. 4a and 4b show FACS histograms for lack of CD14, CD34 and CD45 instained (FIG. 4a ) and unstained (FIG. 4b ) cells.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition, as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “acell” includes “cells”, reference to “a tissue” includes two or moresuch tissues, reference to “a patient” includes two or more suchpatients, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

PMLs of the Invention

The present invention provides a progenitor cell of mesodermal lineage(PML). The PML expresses detectable levels of CD29, CD44, CD73, CD90,CD105 and CD271, but does not express detectable levels of CD14, CD34and CD45.

The PMLs of the invention have numerous advantages. The key advantageswill be summarized here. However, further advantages will becomeapparent from the discussion below.

The PMLs of the invention may advantageously be used to repair damagedtissues in patients. The PMLs are capable of efficiently migrating (orhoming) to a damaged tissue and exerting anti-inflammatory effects inthe tissue. This is discussed in more detail below. One of the mostimportant abilities of the PMLs is to migrate (or home) to injuredsites, which involves chemotaxis. This is based on chemokine-signallingand utilises mechanisms such as rolling, adhesion and transmigration.The anti-inflammatory effects of the PMLs promote survival, repair andregeneration of the neighbouring cells in the damaged tissue. The cellsare also able to exert paracrine effects such as the secretion ofangiogenic, chemotactic and anti-apoptotic factors.

As discussed in more detail below, the PMLs are produced frommononuclear cells (MCs), such as peripheral MCs, taken from a humanindividual. Since the PMLs are produced from MCs, they may be producedeasily (such as from peripheral blood) and may be autologous for thepatient to be treated and thereby avoid the risk of immunologicalrejection by the patient.

It is possible, in principle, to produce an unlimited number of PMLsfrom a single individual, since various samples of MCs (i.e. varioussamples of blood) may be obtained. It is certainly possible to producevery large numbers of PMLs from a single individual. The PMLs of theinvention can therefore be made in large numbers.

The PMLs of the invention are produced in clinically relevantconditions, for instance in the absence of trace amounts of endotoxinsand other environmental contaminants, as well as animal products such asfetal calf serum. This makes the PMLs of the invention particularlysuitable for administration to patients.

Since the PMLs of the invention are produced from MCs, they aresubstantially homologous and may be autologous. They also avoiddonor-to-donor variation, which frequently occurs with mesenchymal stemcells (MSCs). Numerous populations of PMLs of the invention can beproduced from a single sample taken from the patient before any othertherapy, such as chemotherapy or radiotherapy, has begun. Therefore, thePMLs of the invention can avoid any of the detrimental effects of thosetreatments.

The PMLs of the invention can be made quickly. PMLs can be produced fromMCs in less than 30 days, such as in about 22 days.

The production of PMLs from MCs avoids the moral and ethicalimplications involved with using mesenchymal stem cells (MSCs) derivedfrom human embryonic stem cells (hESCs).

The PMLs of the invention are typically produced from human MCs. ThePMLs of the invention are therefore typically human.

The PMLs of the invention can be identified as progenitor cells ofmesodermal lineage using standard methods known in the art, includingexpression of lineage restricted markers, structural and functionalcharacteristics. The PMLs will express detectable levels of cell surfacemarkers known to be characteristic of progenitor cells of mesodermallineage. In particular, in addition to the markers discussed in moredetail below, the PMLs may express α-smooth muscle actin, collagen typeI α-chain, GATA6, Mohawk, and vimentin (Sági B et al Stem Cells Dev.2012 Mar. 20; 21(5):814-28).

The PMLs of the invention are capable of successfully completingdifferentiation assays in vitro to confirm that they are of mesodermallineage. Such assays include, but are not limited to, adipogenicdifferentiation assays, osteogenic differentiation assays and neurogenicdifferentiation assays (Zaim M et al Ann Hematol. 2012 August;91(8):1175-86).

The PMLs of the invention are not stem cells. In particular, they arenot mesenchymal stem cells (MSCs). They are terminally differentiated.Although they can be forced under the right conditions in vitro todifferentiating, for instance into cartilage or bone cells, they do notdifferentiate in vivo. The PMLs of the invention have their effects bymigrating to the damaged tissue and exerting paracrine signalling in thedamaged tissue. In particular, the PMLs are preferably capable ofinducing anti-flammatory effects in the damaged tissue. This isdiscussed in more detail below.

The PMLs of the invention are typically characterised by aspindle-shaped morphology. The PMLs are typically fibroblast-like, i.e.they have a small cell body with a few cell processes that are long andthin. The cells are typically from about 10 to about 20 μm in diameter.

The PMLs of the invention are distinguished from known PMLs via theirmarker expression pattern. The PMLs of the invention express detectablelevels of CD29, CD44, CD73, CD90, CD105 and CD271. The PMLs of theinvention may overexpress one or more of, such as all of, CD29, CD44,CD73, CD90, CD105 and CD271. The PMLs of the invention overexpress oneor more of CD29, CD44, CD73, CD90, CD105 and CD271 if they express morethan other PMLs and/or MSCs. The PMLs of the invention do not expressdetectable levels of CD14, CD34 and CD45.

CD29 (Beta 4 integrin) is an integrin unit associated with very lateantigen receptors. It is known to conjoin with alpha-3 subunit to createan α3β1 complex that reacts with netrin-1 and reelin.

CD44 is a cell-surface glycoprotein involved in cell-cell interactions,cell adhesion and migration. In humans, the CD44 antigen is encoded bythe CD44 gene on Chromosome 11.

CD73, also known as ecto-5′-nucleotidase (ecto-5′-NT, EC 3.1.3.5), is aglycosylphosphatidylinositol-linked 70-kDa cell surface ectoenzyme foundin many types of human and mouse cancers.

CD90 (or Thy-1) is a 25-37 kDa heavily N-glycosylated,glycophosphatidylinositol (GPI) anchored conserved cell surface proteinwith a single V-like immunoglobulin domain. It was originally discoveredas a thymocyte antigen.

CD105 (or Endoglin) is a type I membrane glycoprotein located on cellsurfaces and is part of the TGF beta receptor complex.

CD271, also known as low affinity nerve growth factor receptor (LNGFR)or p75NTR, belongs to the low affinity neurotrophin receptor and tumornecrosis factor receptor superfamily.

CD14 is a component of the innate immune system and exists in two forms.It is either anchored into the membrane by aglycosylphosphatidylinositol tail (mCD14) or it appears in a solubleform (sCD14). Soluble CD14 either appears after shedding of mCD14 (48kDa) or is directly secreted from intracellular vesicles (56 kDa).

CD34 is a cell surface glycoprotein and functions as a cell-celladhesion factor. For instance, it mediates the attachment of stem cellsto bone marrow extracellular matrix or directly to stromal cells.

CD45 is a protein tyrosine phosphatase (PTP) located in hematopoieticcells except ethrocytes and platelets. CD45 is also called the commonleukocyte antigen, T220 and B220 in mice. The protein tyrosine kinasesconstitute a family of receptor-like and cytoplasmic inducing enzymesthat catalyze the dephosphorylation of phosphostyrosine residues and arecharacterized by homologous catalytic domains.

Standard methods known in the art may be used to determine thedetectable expression, low expression or lack thereof of the variousmarkers discussed above (and below). Suitable methods include, but arenot limited to, immunocytochemistry, immunoassays, flow cytometry, suchas fluorescence activated cells sorting (FACS), and polymerase chainreaction (PCR), such as reverse transcription PCR (RT-PCR). Suitableimmunoassays include, but are not limited to, Western blotting,enzyme-linked immunoassays (ELISA), enzyme-linked immunosorbent spotassays (ELISPOT assays), enzyme multiplied immunoassay techniques,radioallergosorbent (RAST) tests, radioimmunoassays, radiobinding assaysand immunofluorescence. Western blotting, ELISAs and RT-PCR are allquantitative and so can be used to measure the level of expression ofthe various markers if present. The use of FACS is disclosed in theExample. Antibodies and fluorescently-labelled antibodies for all of thevarious markers discussed herein are commercially-available.

The PMLs of the invention are preferably capable of migrating to aspecific damaged tissue in a patient. In other words, when the cells areadministered to a patient having a damaged tissue, the cells are capableof migrating (or homing) to the damaged tissue. This is advantageousbecause it means that the cells can be infused via standard routes, forinstance intravenously, and will then target the site of damage. Thecells do not have to be delivered to the damaged tissue. The damage maybe due to injury or disease as discussed in more detail below.

The ability of the PMLs of the invention to migrate to damaged tissuemay be measured using standard assays known in the art. Suitable methodsinclude, but are not limited to, genomic reverse transcriptionpolymerase chain reaction (RT-PCR with or without reporter genes) andlabelling techniques.

RT-PCR is the most straightforward and simple means to trace the PMLs ofthe invention within a patient. A transduced transgene or individualdonor markers can be used for this purpose and transplantedcell-specific signals have been obtained in several patient studies. Theresults are generally semi-quantitative.

Alternatively, the PMLs of the invention may be stained with a dye ofinterest, such as a fluorescent dye, and may be monitored in the patientvia the signal from the dye. A specific method of such labelling isdisclosed in the Example.

Migration (or homing) is typically determined by measuring the number ofcells that arrive at the damaged tissue. It may also be measuredindirectly by observing the numbers of cells that have accumulated inthe lungs (rather than the damaged tissue).

The PMLs of the invention which are capable of migrating to a specific,damaged tissue in a patient preferably (a) express detectable levels of,or overexpress, C-X-C chemokine receptor type 1 (CXCR1) and/or (b)express detectable levels of, or overexpress, CXCR2. The PMLs of theinvention more preferably express detectable levels of, or overexpress,CXCR1 and CXCR2. Damaged tissues release a variety of solubleinflammatory factors, such as macrophage migration inhibitory factor(MIF) and interleukin-8, and these factors may attract the PMLs of theinvention (and other inflammatory cells) to the damaged tissue thoughbinding to binding CXCR1 and/or CXCR2.

The PMLs of the invention overexpress CXCR1 and/or CXCR2 if they producemore CXCR1 and/or CXCR2 that other PMLs and/or MSCs. The expression ofCXCR1 and/or CXCR2 may be measured as discussed above. Theretinal-homing cells of the invention do not express detectable levelsof CXCR1 and CXCR2. This is discussed in more detail below.

The specific, damaged tissue to which the PMLs of the invention arecapable of migrating is preferably cardiac tissue, retinal tissue orbone tissue. The retinal tissue is preferably the macula.

If the specific, damaged tissue is heart tissue or bone tissue, the PMLsof the invention preferably express detectable levels of, oroverexpress, C-X-C chemokine receptor type 4 (CXCR4). The PMLs of theinvention overexpress CXCR4 if they express more CXCR4 that other PMLsand/or MSCs. If the specific, damaged tissue is heart tissue or bonetissue, the PMLs of the invention more preferably express detectablelevels of, or overexpress, (a) CXCR1 and CXCR4; (b) CXCR2 and CXCR4; or(c) CXCR1, CXCR2 and CXCR4. The expression of CXCR4 may be measured asdiscussed above.

Damaged heart tissue releases inflammatory chemokines and cytokines,such as stromal cell-derived factor-1 (SDF-1), interleukin-8 (IL-8),tumor necrosis factor-alpha (TNF-alpha), granulocyte-colony-stimulatingfactor (G-CSF), vascular endothelial growth factor (VEGF) and hepatocytegrowth factor (HGF). In addition, myocardial infarct increases thelevels of VEGF and erythropoietin (EPO). CXCR4 binds to its ligand SDF-1and so PMLs of the invention expressing CXCR4 will migrate towards thegradient of SDF-1 generated by the damaged heart tissue. Other damagedtissues, such as bone, also release SDF-1.

If the specific, damaged tissue is retinal tissue, such as the macula,the PMLs of the invention preferably express detectable levels of CXCR4,vascular endothelial growth factor (VEGF), transforming growth factorbeta 1 (TGF-beta 1), insulin-like growth factor-1 (IGF-1), fibroblastgrowth factor (FGF), tumour necrosis factor alpha (TNF-alpha),interferon gamma (IFN-gamma), interleukin-1 alpha (IL-1 alpha), CXCL12,CD109, CD119, nuclear factor kappa-light-chain-enhancer of activated Bcells (NFkappa B), CD140a, CD140b, CD221, CD222, CD304, CD309 and CD325.The retinal-homing PMLs of the invention preferably overexpress one ormore of, or even all of, these factors. The PMLs overexpress thesefactors if they express more of the factors than other PMLs and/or MSCs.Quantitative assays for cell markers are described above.

Retinal-homing PMLs of the invention preferably also express detectablelevels of pigment epithelium derived factor (PEDF) or overexpress PEDF.The detectable expression of these markers may be measured as discussedabove. The PMLs of the invention overexpress PEDF if they express morePEDF than other PMLs and/or mesenchymal stem cells (MSCs).

If the specific, damaged tissue is bone tissue, the PMLs of theinvention preferably express detectable levels of TGF-beta 3, bonemorphogenetic protein-6 (BMP-6), SOX-9, Collagen-2, CD117 (c-kit),chemokine (C-C motif) ligand 12 (CCL12), CCL7, interleukin-8 (IL-8),platelet-derived growth factor-A (PDGF-A), PDGF-B, PDGF-C, PDGF-D,macrophage migration inhibitory factor (MIF), IGF-1, hepatocyte growthfactor (HGF), PDGF-Rα, PDGF-Rβ, CXCR4, C-C chemokine receptor type 1(CCR1), IGF-1 receptor (IGF-1R), hepatocyte growth factor receptor(HGFR), CXCL12 and NFkappaB. The bone-homing PMLs of the inventionpreferably overexpress one or more of, or even all of, these factors.The PMLs overexpress these factors if they express more of the factorsthan other PMLs and/or MSCs. The detectable expression of these markersmay be measured as discussed above.

The PMLs of the invention are preferably capable of havinganti-inflammatory effects in a damaged tissue of a patient. The abilityof the PMLs of the invention to have anti-inflammatory effects may alsobe measured using standard assays known in the art. Suitable methodsinclude, but are not limited to, enzyme-linked immunosorbent assays(ELISAs) for the secretion of cytokines, enhanced mixed leukocytereactions and up-regulation of co-stimulatory molecules and maturationmarkers, measured by flow cytometry. Specific methods that may be usedare disclosed in the Example. The cytokines measured are typicallyinterleukins, such as interleukin-8 (IL-8), selectins, adhesionmolecules, such as Intercellular Adhesion Molecule-1 (ICAM-1), andchemoattractant proteins, such as monocyte chemotactic protein-1 (MCP-1)and tumour necrosis factor alpha (TNF-alpha). Assays for these cytokinesare commercially-available.

Anti-inflammatory PMLs preferably express detectable levels of CD120a(tumour-necrosis factor (TNF)-alpha Receptor 1), CD120b (TNF-alphaReceptor 2), CD50 (Intercellular Adhesion Molecule-3, ICAM-3), CD54(ICAM-1), CD58 (Lymphocyte function-associated antigen-1, LFA-1), CD62E(E-selectin), CD62L (L-selectin), CD62P (P-selectin), CD106 (Vascularcell adhesion protein, VCAM-1), CD102 (ICAM-2), CD166 (Activatedleukocyte cell adhesion molecule), CD104 (Beta 4 integrin), CD123(Interleukin-3 Receptor), CD124 (Interleukin-4 Receptor), CD126(Interleukin-6 Receptor), CD127 (Interleukin-7 Receptor) and fibroblastgrowth factor receptor (FGFR). Anti-inflammatory PMLs preferablyoverexpress one or more of, or even all of, these factors. The PMLsoverexpress these factors if they express more of the one or morefactors than other PMLs and/or MSCs. The detectable expression of thesemarkers may be measured as discussed above.

The PMLs of the invention are more preferably capable of migrating to adamaged tissue in a patient and having anti-inflammatory effects in thedamaged tissue. This allows the damage to be repaired effectively andreduces the number of cells that need to be administered.

The PMLs of the invention will express a variety of different othermarkers over and above those discussed above. Some of these will assistthe PMLs will their ability to migrate to a damaged tissue and haveanti-inflammatory effects once there. Any of the PMLs of the inventionmay further express detectable levels of one or more of (i) insulin-likegrowth factor-1 (IGF-1), (ii) IGF-1 receptor; (iii) C-C chemokinereceptor type 1 (CCR1), (iv) stromal cell-derived factor-1 (SDF-1), (v)hypoxia-inducible factor-1 alpha (HIF-1 alpha), (vi) Akt1 and (vii)hepatocyte growth factor (HGF) and/or granulocyte colony-stimulatingfactor (G-CSF).

IGF-1 receptors promote migration capacity towards an IGF-1 gradient.One of the mechanisms by which IGF-1 increases migration is byup-regulating CXCR4 on the surface of the cells, which makes them moresensitive to SDF-1 signaling. This is discussed above.

CCR1 is the receptor for CCL7 (previously known as MCP3) increaseshoming and engraftment capacity of MSCs (and so would be expected tohave the same effect for the PMLs of the invention) and can increase thecapillary density in injured myocardium through paracrine signalling.

HIF-1 alpha activates pathways that increase oxygen delivery and promoteadaptive pro-survival responses. Among the many target genes of HIF-1alpha are erythropoietin (EPO), endothelin and VEGF (with its receptorFlk-1). PMLs that express or overexpress HIF-1alpha will haveupregulated expression of paracrine stimuli of for example severalvasculogenic growth factors that may promote a more therapeutic subtype.As described in more detail below, the PMLs of the invention can bepreconditioned into a more therapeutic subtype by culturing them underhypoxic conditions (less than 20% oxygen), such as for example about 2%or about 0% oxygen.

Akt1 is an intracellular serine/threonine protein kinase that plays akey role in multiple cellular processes such as glucose metabolism, cellproliferation, apoptosis, transcription and cell migration.Overexpression of Akt1 has been shown to prevent rat MSCs fromundergoing apoptosis and will have the same effect in the PMLs of theinvention. Protection from apoptosis will enhance the therapeutic effectof the PMLs.

The overexpression of HGF by MSCs has been shown to preventpost-ischemic heart failure by inhibition of apoptosis viacalcineurin-mediated pathway and angiogenesis. HGF and G-CSF exhibitsynergistic effects in this regard. MSCs that have a high expression ofHGF and its receptor c-met also have an increased migratory capacityinto the damaged tissue, achieved through hormonal, paracrine andautocrine signaling. The same will be true for the PMLs of the inventionexpressing HGF and/or G-CSF.

The PMLs may overexpress one or more of (i) to (vii) defined above. ThePMLs of the invention overexpress one or more of (i) to (vii) if theyexpress more than other PMLs and/or than mesenchymal stem cells (MSCs).Quantitative assays for cell markers are described above. The detectableexpression of these markers and their level of expression may bemeasured as discussed above.

Any of the PMLs of the invention may express detectable levels of one ormore of (i) vascular endothelial growth factor (VEGF), (ii) transforminggrowth factor beta (TGF-beta), (iii) insulin-like growth factor-1(IGF-1), (iv) fibroblast growth factor (FGF), (v) tumour necrosis factoralpha (TNF-alpha), (vi) interferon gamma (IFN-gamma) and (vii)interleukin-1 alpha (IL-1 alpha). Conditioned medium from cellsoverexpressing VEGF has been shown to alleviate heart failure in ahamster model. Hence, the PMLs of the invention which express oroverexpress VEGF will have the same effect of damaged cardiac tissue.

The PMLs may overexpress one or more of (i) to (vii). The PMLs of theinvention overexpress one or more of (i) to (vii) if they express morethan other PMLs and/or than mesenchymal stem cells (MSCs). Quantitativeassays for cell markers are described above. The detectable expressionof these markers and their level of expression may be measured asdiscussed above.

In both sets of definitions of (i) to (vii) given above, any combinationof one or more of (i) to (vii) may be expressed or overexpressed. Forinstance, for each definition of (i) to (vii), the PMLs may expressdetectable levels of, or overexpress, (i); (ii); (iii); (iv); (v); (vi);(vii); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and(vi); (i) and (vii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii)and (vi); (ii) and (vii); (iii) and (iv); (iii) and (v); (iii) and (vi);(iii) and (vii); (iv) and (v); (iv) and (vi); (iv) and (vii); (v) and(vi); (v) and (vii); (vi) and (vii); (i), (ii) and (iii); (i), (ii) and(iv); (i), (ii) and (v); (i), (ii) and (vi); (i), (ii) and (vii); (i,),(iii) and (iv); (i), (iii) and (v); (i), (iii) and (vi); (i), (iii) and(vii); (i), (iv) and (v); (i), (iv) and (vi); (i), (iv) and (vii); (i),(v) and (vi); (i), (v) and (vii); (i), (vi) and (vii); (ii), (iii) and(iv); (ii), (iii) and (v); (ii), (iii) and (vi); (ii), (iii) and (vii);(ii), (iv) and (v); (ii), (iv) and (vi); (ii), (iv) and (vii); (ii), (v)and (vi); (ii), (v) and (vii); (ii), (vi) and (vii); (iii), (iv) and(v); (iii), (iv) and (vi); (iii), (iv) and (vii); (iii), (v) and (vi);(iii), (v) and (vii); (iii), (vi) and (vii); (iv), (v) and (vi); (iv),(v) and (vii); (iv), (vi) and (vii); (v), (vi) and (vii); (i), (ii),(iii) and (iv); (i), (ii), (iii) and (v); (i), (ii), (iii) and (vi);(i), (ii), (iii) and (vii); (i), (ii), (iv) and (v); (i), (ii), (iv) and(vi); (i), (ii), (iv) and (vii); (i), (ii), (v) and (vi); (i), (ii), (v)and (vii); (i), (ii), (vi) and (vii); (i), (iii), (iv) and (v); (i),(iii), (iv) and (vi); (i), (iii), (iv) and (vii); (i), (iii), (v) and(vi); (i), (iii), (v) and (vii); (i), (iii), (vi) and (vii); (i), (iv),(v) and (vi); (i), (iv), (v) and (vii); (i), (iv), (vi) and (vii); (i),(v), (vi) and (vii); (ii), (iii), (iv) and (v); (ii), (iii), (iv) and(vi); (ii), (iii), (iv) and (vii); (ii), (iii), (v) and (vi); (ii),(iii), (v) and (vii); (ii), (iii), (vi) and (vii); (ii), (iv), (v) and(vi); (ii), (iv), (v) and (vii); (ii), (iv), (vi) and (vii); (ii), (v),(vi) and (vii); (iii), (iv), (v) and (vi); (iii), (iv), (v) and (vii);(iii), (iv), (vi) and (vii); (iii), (v), (vi) and (vii); (iv), (v), (vi)and (vii); (i), (ii), (iii), (iv) and (v); (i), (ii), (iii), (iv) and(vi); (i), (ii), (iii), (iv) and (vii); (i), (ii), (iii), (v) and (vi);(i), (ii), (iii), (v) and (vii); (i), (ii), (iii), (vi) and (vii); (i),(ii), (iv), (v) and (vi); (i), (ii), (iv), (v) and (vii); (i), (ii),(iv), (vi) and (vii); (i), (ii), (v), (vi) and (vii); (i), (iii), (iv),(v) and (vi); (i), (iii), (iv), (v) and (vii); (i), (iii), (iv), (vi)and vii); (i), (iii), (v), (vi) and (vii); (i), (iv), (v), (vi) and(vii); (ii), (iii), (iv), (v) and (vi); (ii), iii), (iv), (v) and (vii);(ii), (iii), (iv), (vi) and (vii); (ii), (iii), (v), (vi) and (vii);(ii), (iv), (v), (vi) and (vii); (iii), (iv), (v), (vi) and vii); (i),(ii), (iii), (iv), (v) and (vi); (i), (ii), (iii), (iv), (v) and (vii);(i), (ii), (iii), (iv), (vi) and (vii); (i), (ii), (iii), (v), (vi) and(vii); (i), (ii), (iv), (v), (vi) and (vii); (i), (iii), (iv), (v), (vi)and (vii); (ii), (iii), (iv), (v), (vi) and (vii); or (i), (ii), (iii),(iv), (v), (vi) and (vii). The combinations for each definition of (i)to (vii) are independently selectable from this list.

In addition to any of the markers discussed above, the PMLs of theinvention preferably also express detectable levels of, or overexpress,LIF and/or platelet-derived growth factor (PDGF) receptors. The PDGFreceptors are preferably PDGF-A receptors and/or PSDGF-B receptors. MSCsthat have high expression of these receptors can migrate effectivelyinto areas in which platelets have been activated, such as wounds andthrombotic vessels. The same will be true of PMLs expressing oroverexpressing the receptors.

The PMLs of the invention are preferably autologous. In other words, thecells are preferably derived from the patient into which the cells willbe administered. Alternatively, the PMLs are preferably allogeneic. Inother words, the cells are preferably derived from a patient that isimmunologically compatible with the patient into which the cells will beadministered.

A PML of the invention may be isolated, substantially isolated, purifiedor substantially purified. The PML is isolated or purified if it iscompletely free of any other components, such as culture medium, othercells of the invention or other cell types. The PML is substantiallyisolated if it is mixed with carriers or diluents, such as culturemedium, which will not interfere with its intended use. Alternatively,the PML of the invention may be present in a growth matrix orimmobilized on a surface as discussed below.

PMLs of the invention may be isolated using a variety of techniquesincluding antibody-based techniques. Cells may be isolated usingnegative and positive selection techniques based on the binding ofmonoclonal antibodies to those surface markers which are present on thePML (see above). Hence, the PMLs may be separated using anyantibody-based technique, including fluorescent activated cell sorting(FACS) and magnetic bead separation.

As discussed in more detail below, the PMLs may be treated ex vivo. Thusthe cells may be loaded or transfected with a therapeutic or diagnosticagent and then used therapeutically in the methods of the invention.

Population of the Invention

The invention also provides a population of two or more PMLs of theinvention. Any number of cells may be present in the population. Thepopulation of the invention preferably comprises at least about 5×10⁵PMLs of the invention. The population more preferably comprises at leastabout 1×10⁶, at least about 2×10⁶, at least about 5×10⁶, at least about1×10⁷, at least about 2×10⁷, at least about 5×10⁷, at least about 1×10⁸or at least about 2×10⁸ PMLs of the invention. In some instances, thepopulation may comprise at least about 1.0×10⁷, at least about 1.0×10⁸,at least about 1.0×10⁹, at least about 1.0×10¹⁰, at least about 1.0×10¹¹or at about least 1.0×10¹² PMLs of the invention or even more.

The populations of the invention are advantageous for therapy asdiscussed below. This ability to produce populations comprising largenumbers of PMLs of the invention is one of the key advantages of theinvention. The invention allows the treatment of patients with apopulation of cells of which most, if not all, migrate efficiently tothe tissue of interest and have anti-inflammatory effects once there.This allows the use of a low cell-dose and avoids off-target sideeffects and volume-related side effects.

The population of the invention may comprise other cells in addition tothe PMLs of the invention. However, at least 70% of the cells in thepopulation are preferably PMLs of the invention. More preferably, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 97%, at least about 98% or at least about 99% of thecells in the population are PMLs of the invention.

The population of the invention is preferably homologous. In otherwords, all of the PMLs in the population are preferably genotypicallyand phenotypically identical. The population is preferably autologous orallogeneic as defined above.

However, the population can also be semi-allogeneic. Semi-allogeneicpopulations are typically produced from mononuclear cells from two ormore patients that are immunologically compatible with the patient intowhich the population will be administered. In other words, all of thecells in the population are preferably genetically identical orsufficiently genetically identical that the population isimmunologically compatible with the patient into which the populationwill be administered. Since the PMLs of the invention may be derivedfrom a patient, they may be autologous with the patient to be treated(i.e. genetically identical with the patient or sufficiently geneticallyidentical that they are compatible for administration to the patient).

The population of the invention may be isolated, substantially isolated,purified or substantially purified. A population is isolated or purifiedif it is completely free of any other components, such as culture mediumand other cells. A population is substantially isolated if it is mixedwith carriers or diluents, such as culture medium, which will notinterfere with its intended use. Other carriers and diluents arediscussed in more detail below. A substantially isolated orsubstantially purified population does not comprise cells other than thePMLs of the invention. In some embodiments, the population of theinvention may be present in a growth matrix or immobilized on a surfaceas discussed below.

The population is typically cultured in vitro. Techniques for culturingcells are well known to a person skilled in the art. The cells are maybe cultured under standard conditions of 37° C., 5% CO₂ in mediumwithout serum. The cells are preferably cultured under low oxygenconditions as discussed in more detail below. The cells may be culturedin any suitable flask or vessel, including wells of a flat plate such asa standard 6 well plate. Such plates are commercially available fromFisher scientific, VWR suppliers, Nunc, Starstedt or Falcon. The wellstypically have a capacity of from about 1 ml to about 4 ml.

The flask, vessel or wells within which the population is contained orcultured may be modified to facilitate handling of the PMLs. Forinstance, the flask, vessel or wells may be modified to facilitateculture of the cells, for instance by including a growth matrix. Theflask, vessel or wells may be modified to allow attachment of the PMLsor to allow immobilization of the PMLs onto a surface. One or moresurfaces may be coated with extracellular matrix proteins such aslaminin or collagen or any other capture molecules that bind to thecells and immobilize or capture them on the surface(s).

The population may be modified ex vivo using any of the techniquesdescribed herein. For instance, the population may be transfected orloaded with therapeutic or diagnostics agents. The population may thenbe used in the methods of treatment discussed in more detail below.

Method of Producing a PML of the Invention

The invention also provides a method for producing a population of theinvention, i.e. a population of two or more PMLs of the invention. Themethod involves culturing mononuclear cells (MCs) under conditions whichinduce the MCs to differentiate into PMLs. The method then involvesharvesting and culturing the PMLs which:

-   -   (a) express detectable levels of CD29, CD44, CD73, CD90, CD105        and CD271 and do not express detectable levels of CD14, CD34 and        CD45;    -   (b) express detectable levels of CD29, CD44, CD73, CD90, CD105,        CD271 and CXCR1 and do not express detectable levels of CD14,        CD34 and CD45;    -   (c) express detectable levels of CD29, CD44, CD73, CD90, CD105,        CD271 and CXCR2 and do not express detectable levels of CD14,        CD34 and CD45;    -   (d) express detectable levels of CD29, CD44, CD73, CD90, CD105,        CD271, CXCR1 and CXCR2 and do not express detectable levels of        CD14, CD34 and CD45;    -   (e) express detectable levels of CD29, CD44, CD73, CD90, CD105,        CD271, CXCR1 and CXCR4 and do not express detectable levels of        CD14, CD34 and CD45 (these cells are heart-homing and        bone-homing PMLs);    -   (f) express detectable levels of CD29, CD44, CD73, CD90, CD105,        CD271, CXCR2 and CXCR4 and do not express detectable levels of        CD14, CD34 and CD45 (these cells are heart-homing and        bone-homing PMLs);    -   (g) express detectable levels of CD29, CD44, CD73, CD90, CD105,        CD271, CXCR1, CXCR2 and CXCR4 and do not express detectable        levels of CD14, CD34 and CD45 (these cells are heart-homing and        bone-homing PMLs);    -   (h) express detectable levels of CD29, CD44, CD73, CD90, CD105,        CD271, CXCR4, vascular endothelial growth factor (VEGF),        transforming growth factor beta 1 (TGF-beta 1), insulin-like        growth factor-1 (IGF-1), fibroblast growth factor (FGF), tumour        necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma),        interleukin-1 alpha (IL-1 alpha), CXCL12, CD109, CD119, nuclear        factor kappa-light-chain-enhancer of activated B cells (NFkappa        B), CD140a, CD140b, CD221, CD222, CD304, CD309 and CD325 and do        not express detectable levels of CD14, CD34 and CD45 (these        cells are retinal-homing PMLs); or    -   (i) express detectable levels of CD29, CD44, CD73, CD90, CD105,        CD271, TGF-beta 3, bone morphogenetic protein-6 (BMP-6), SOX-9,        Collagen-2, CD117 (c-kit), chemokine (C-C motif) ligand 12        (CCL12), CCL7, interleukin-8 (IL-8), platelet-derived growth        factor-A (PDGF-A), PDGF-B, PDGF-C, PDGF-D, macrophage migration        inhibitory factor (MIF), IGF-1, hepatocyte growth factor (HGF),        PDGF-Rα, PDGF-Rβ, CXCR4, C-C chemokine receptor type 1 (CCR1),        IGF-1 receptor (IGF-1R), hepatocyte growth factor receptor        (HGFR), CXCL12 and NFkappaB and do not express detectable levels        of CD14, CD34 and CD45 (these cells are bone-homing PMLs).

The harvested cells may overexpress any of the factors as describedabove with reference to the cells of the invention. In addition to anyone of (a) to (i) above, the method preferably involves harvesting andculturing PMLs which:

-   -   (j) express detectable levels of CD120a (tumour-necrosis factor        (TNF)-alpha Receptor 1), CD120b (TNF-alpha Receptor 2), CD50        (Intercellular Adhesion Molecule-3, ICAM-3), CD54 (ICAM-1), CD58        (Lymphocyte function-associated antigen-1, LFA-1), CD62E        (E-selectin), CD62L (L-selectin), CD62P (P-selectin), CD106        (Vascular cell adhesion protein, VCAM-1), CD102 (ICAM-2), CD166        (Activated leukocyte cell adhesion molecule), CD104 (Beta 4        integrin), CD123 (Interleukin-3 Receptor), CD124 (Interleukin-4        Receptor), CD126 (Interleukin-6 Receptor), CD127 (Interleukin-7        Receptor) and fibroblast growth factor receptor (FGFR);    -   (k) express detectable levels of one or more of (i) insulin-like        growth factor-1 (IGF-1), (ii) IGF-1 receptor; (iii) C-C        chemokine receptor type 1 (CCR1), (iv) stromal cell-derived        factor-1 (SDF-1), (v) hypoxia-inducible factor-1 alpha (HIF-1        alpha), (vi) Akt1 and (vii) hepatocyte growth factor (HGF)        and/or granulocyte colony-stimulating factor (G-CSF);    -   (l) overexpress one or more of (i) to (vii) in (k);    -   (m) express detectable levels of one or more of (i) vascular        endothelial growth factor (VEGF), (ii) transforming growth        factor beta (TGF-beta), (iii) insulin-like growth factor-1        (IGF-1), (iv) fibroblast growth factor (FGF), (v) tumour        necrosis factor alpha (TNF-alpha), (vi) interferon gamma        (IFN-gamma) and (vii) interleukin-1 alpha (IL-1 alpha)    -   (n) overexpress one or more of (i) to (vii) in (m).

Mononuclear cells (MCs) and methods of isolating them are known in theart. The MCs may be primary MCs isolated from bone marrow. The MCs arepreferably peripheral blood MCs (PBMCs), such as lymphocytes, monocytesand/or macrophages. PBMCs can be isolated from blood using a hydrophilicpolysaccharide, such as Ficoll®. For instance, PBMCs may be isolatedfrom blood using Ficoll-Paque® (a commercially-available density medium)as disclosed in the Example.

Before they are cultured, the MCs may be exposed to a mesenchymal stemcell enrichment cocktail. The cocktail preferably comprises antibodiesthat recognise CD3, CD14, CD19, CD38, CD66b (which are present onunwanted cells) and a component of red blood cells. Such a cocktailcross links unwanted cells with red blood cells forming immunorosetteswhich may be removed from the wanted MCs. A preferred cocktail isRosetteSep®.

Conditions suitable for inducing MCs to differentiate into mesenchymalcells (tissue mainly derived from the mesoderm) are known in the art.For instance, suitable conditions are disclosed in Capelli, C., et al.(Human platelet lysate allows expansion and clinical grade production ofmesenchymal stromal cells from small samples of bone marrow aspirates ormarrow filter washouts. Bone Marrow Transplantation, 2007. 40: p.785-791). These conditions may also be used to induce MCs todifferentiate into PMLs in accordance with the invention.

The method preferably comprises culturing MCs with plasma lysate toinduce the MCs to differentiate into PMLs. Platelet lysate refers to thecombination of natural growth factors contained in platelets that hasbeen released through lysing those platelets. Lysis can be accomplishedthrough chemical means (i.e. CaCl₂)), osmotic means (use of distilledH₂O) or through freezing/thawing procedures. Platelet lysate can bederived from whole blood as described in U.S. Pat. No. 5,198,357.Platelet lysate is preferably prepared as described in the Example. Theplasma lysate is preferably human plasma lysate.

In a preferred embodiment, step (a) of the method of the inventioncomprises culturing MCs in a medium comprising platelet lysate forsufficient time to induce the MCs to differentiate into progenitor cellsof mesodermal lineage. The sufficient time is typically from about 15 toabout 25 days, preferably about 22 days. The medium preferably comprisesabout 20% or less platelet lysate by volume, such as about 15% or lessby volume or about 10% or less by volume. The medium preferablycomprises from about 5% to about 20% of platelet lysate by volume, suchas from about 10% to about 15% by volume. The medium preferablycomprises about 10% of platelet lysate by volume.

In another preferred embodiment, step (a) of the method of the inventioncomprises exposing MCs to a mesenchymal enrichment cocktail and thenculturing the MCs in a medium comprising platelet lysate for sufficienttime to induce the MCs to differentiate into progenitor cells ofmesodermal lineage. The sufficient time is typically from about 15 toabout 25 days, preferably about 22 days.

In step (a), the medium is preferably Minimum Essential Medium (MEM).MEM is commercially available from various sources includingSigma-Aldrich. The medium preferably further comprises one or more ofheparin, L-glutamine and penicillin/streptavidin (P/S). The L-glutaminemay be replaced with GlutaMAX® (which is commercially-available fromLife Technologies).

As discussed above, some of the PMLs of the invention express detectablelevels of CXCR4. Expression of CXCR4 is cytokine-dependent and isincreased when cells are exposed to stem cell factor (SCF),interleukin-6 (IL-6), Flt-3 ligand, hepatocyte growth factor (HGF) andIL-3. The medium may comprise one or more of (i) SCF, (ii) IL-6, (iii)Flt-3 ligand, (iv) hepatocyte growth factor and (v) IL-3, such as (i);(ii); (iii); (iv); (v); (i) and (ii); (i) and (iii); (i) and (iv); (i)and (v); (ii) and (iii); (ii) and (iv); (ii) and (v); (iii) and (iv);(iii) and (v); (iv) and (v); (i), (ii) and (iii); (i), (ii) and (iv);(i), (ii) and (v); (i), (iii) and (iv); (i), (iii) and (v); (i), (iv)and (v); (ii), (iii) and (iv); (ii), (iii) and (v); (ii), (iv) and (v);(iii), (iv) and (v); or (i), (ii), (iii), (iv) and (v). Any of (i) to(v) may be present at from about from about 10 to about 150 ng/ml.

Step (a) preferably comprises culturing the MCs under conditions whichallow the PMLs to adhere. Suitable conditions are discussed in moredetail above.

In step (a), the MCs are preferably cultured under low oxygenconditions. The MCs are preferably cultured at less than about 20%oxygen (O₂), such as less than about 19%, less than about 18%, less thanabout 17%, less than about 16%, less than about 15%, less than about14%, less than about 13%, less than about 12%, less than about 11%, lessthan about 10%, less than about 9%, less than about 8%, less than about7%, less than about 6%, less than about 5%, less than about 4%, lessthan about 3%, less than about 2% or less than about 1% oxygen (O₂). TheMCs are preferably cultured at from about 0% to about 19% O₂, such asfrom about 1% to about 15% O₂, from about 2% to about 10% 02 or fromabout 5% to about 8% O₂. The MCs are most preferably cultured at about0% 02. The figures for % oxygen (or % O₂) quoted above relate to % byvolume of oxygen in the gas supplied to the cells during culture, forinstance by the cell incubator. It is possible that some oxygen may leakinto the incubator or enter when the door is opened.

In step (a), the MCs are most preferably cultured in the presence ofplatelet lysate and under low oxygen conditions. This combination mimicsthe natural conditions in the damaged tissue and so result in healthierand more therapeutically potent cells. Conventional cell culture isperformed in 20% or 21% oxygen (approximately the atmospheric content)but there is no place in the human body that has this oxygen level. Theepithelial cells in the lungs would “see” this oxygen level, but oncethe oxygen is dissolved and leaves the lungs, it decreases to around17%. From there, it decreases even further to about 1-2% in the majorityof the tissues, but being as low as 0.1% in avascular tissues such asthe cartilage in the joints.

In step (b), the method further comprises harvesting and culturing PMLswhich have the necessary marker expression pattern as discussed above.The PMLs having the necessary marker expression pattern may be harvestedusing any antibody-based technique, including fluorescent activated cellsorting (FACS) and magnetic bead separation. FACS is preferred.

Any of the methods for culturing PMLs disclosed in relation to step (a)equally apply to step (b). In particular, the cells are cultured in step(b) in the presence of platelet lysate and under low oxygen conditionsas discussed above in relation to step (a).

As will be clear from the discussion above, the method of the inventionis carried out in clinically relevant conditions, i.e. in the absence oftrace amounts of endotoxins and other environmental contaminants, suchas lipopolysaccharides, lipopeptides and peptidoglycans, etc. This makesthe PMLs of the invention particularly suitable for administration topatients.

The MCs are preferably obtained from a patient or an allogeneic donor.The invention also provides a method for producing a population of theinvention that is suitable for administration to a patient, wherein themethod comprises culturing MCs obtained from the patient underconditions which induce the MCs to differentiate into progenitor cellsof mesodermal lineage and (b) harvesting and culturing those progenitorcells which have an expression pattern as defined above and therebyproducing a population of the invention that is suitable foradministration to the patient. The population will be autologous withthe patient and therefore will not be rejected upon implantation. Theinvention also provides a population of the invention that is suitablefor administration to a patient and is produced in this manner.

Alternatively, the invention provides a method for producing apopulation of the invention that is suitable for administration to apatient, wherein the method comprises culturing MCs obtained from adifferent patient that is immunologically compatible with the patientinto which the cells will be administered under conditions which inducethe MCs to differentiate into progenitor cells of mesodermal lineage and(b) harvesting and culturing those progenitor cells which have anexpression pattern as defined above and thereby producing a populationof the invention that is suitable for administration to the patient. Thepopulation will be allogeneic with the patient and therefore will reducethe chance of rejection upon implantation. The invention also provides apopulation of the invention that is suitable for administration to apatient and is produced in this manner.

Medicaments, Methods and Therapeutic Use

The PMLs of the invention may be used in a method of therapy of thehuman or animal body. Thus the invention provides a PML of the inventionor a population of the invention for use in a method of treatment of thehuman or animal body by therapy. In particular, the invention concernsusing the PMLs of the invention to repair a damaged tissue in a patient.The invention also concerns using the PMLs of the invention to treat acardiac injury or disease, age-related macular degeneration or a boneinjury or disease in the patient.

The invention provides a method of repairing a damaged tissue in apatient, comprising administering to the patient a population of theinvention, wherein the population comprises a therapeutically effectivenumber of cells, and thereby treating the damaged tissue in the patient.The invention also provides a population of the invention for use inrepairing a damaged tissue in the patient. The invention also providesuse of a population of the invention in the manufacture of a medicamentfor repairing a damaged tissue in a patient.

The tissue is preferably derived from the mesoderm. The tissue is morepreferably cardiac tissue, retinal tissue or bone tissue.

The damage to the tissue may be caused by injury or disease. The injuryor disease is preferably a cardiac injury or disease, age-relatedmacular degeneration (AMD) or a bone injury or disease in a patient. Theinvention therefore provides a method of treating a cardiac injury ordisease, age-related macular degeneration or a bone injury or disease ina patient, comprising administering to the patient a population of theinvention, wherein the population comprises a therapeutically effectivenumber of cells, and thereby treating the cardiac injury or disease,age-related macular degeneration or bone injury or disease in thepatient. The invention also provides a population of the invention foruse in treating a cardiac injury or disease, age-related maculardegeneration or a bone injury or disease in a patient. The inventionalso provides use of a population of the invention in the manufacture ofa medicament for treating a cardiac injury or disease, age-relatedmacular degeneration or a bone injury or disease in a patient.

The cardiac injury or disease is preferably selected from myocardialinfarct (MI), left ventricular hypertrophy, right ventricularhypertrophy, emboli, heart failure, congenital heart deficit, heartvalve disease, arrhythmia and myocarditis.

MI increases the levels of VEGF and EPO released by the myocardium.Furthermore, MI is associated with an inflammatory reaction andinfarcted tissue also releases macrophage migration inhibitory factor(MIF), interleukin (IL-6) and KC/Gro-alpha. CCL7 (previously known asMCP3), CXCL1, CXCL2 are significantly upregulated in the heart followingmyocardial infarct (MI) and might be implicated in regulatingengraftment and homing of MSCs to infarcted myocardium.

In a myocardial infarct mice model, IL-8 was shown to highly up-regulategene expression primarily in the first 2 days post-MI. Remarkably, theincreased IL-8 expression was located predominantly in the infarctedarea and the border zone, and only to a far lesser degree in the sparedmyocardium. By activating CXCR2, MIF displays chemokine-like functionsand acts as a major regulator of inflammatory cell recruitment andatherogenesis.

The AMD may be dry AMD or wet AMD. Dry AMD results from atrophy of theretinal pigment epithelial layer below the retina which causes visionloss through loss of photoreceptors (rods and cones) in the central partof the eye. Wet AMD causes vision loss due to abnormal blood vesselgrowth (choroidal neovascularization) in the choriocapillaris, throughBruch's membrane, ultimately leading to blood and protein leakage belowthe macula. Wet AMD is associated with a decrease in the levels ofpigment epithelium derived factor (PEDF) in the macula. The PMLs used inthe treatment of wet AMD preferably express detectable levels of PEDF oroverexpress PEDF.

The bone disease or injury is preferably selected from fracture,Salter-Harris fracture, greenstick fracture, bone spur,craniosynostosis, Coffin-Lowry syndrome, fibrodysplasia ossificansprogressive, fibrous dysplasia, Fong Disease (or Nail-patella syndrome),hypophosphatasia, Klippel-Feil syndrome, Metabolic Bone Disease,Nail-patella syndrome, osteoarthritis, osteitis deformans (or Paget'sdisease of bone), osteitis fibrosa cystica (or Osteitis fibrosa or VonRecklinghausen's disease of bone), osteitis pubis, condensing osteitis(or osteitis condensans), osteitis condensans ilii, osteochondritisdissecans, osteogenesis imperfecta, osteomalacia, osteomyelitis,osteopenia, osteopetrosis, osteoporosis, osteonecrosis, porotichyperostosis, primary hyperparathyroidism, renal osteodystrophy, bonecancer, a bone lesion associated with metastatic cancer, Gorham Stoutdisease, primary hyperparathyroidism, periodontal disease, and asepticloosening of joint replacements. The bone cancer can be Ewing sarcoma,multiple myeloma, osteosarcoma (giant tumour of the bone),osteochondroma or osteoclastoma. The metastatic cancer that results in abone lesion can be breast cancer, prostate cancer, kidney cancer, lungcancer and/or adult T-cell leukemia.

If the damaged tissue is cardiac tissue or bone tissue, the PMLs in thepopulation preferably express detectable levels of CD29, CD44, CD73,CD90, CD105, CD271, CXCR1, CXCR2 and CXCR4 and do not express detectablelevels of CD14, CD34 and CD45. If the damaged tissue is bone tissue, thePMLs in the population more preferably express detectable levels ofCD29, CD44, CD73, CD90, CD105, CD271, TGF-beta 3, bone morphogeneticprotein-6 (BMP-6), SOX-9, Collagen-2, CD117 (c-kit), chemokine (C-Cmotif) ligand 12 (CCL12), CCL7, interleukin-8 (IL-8), platelet-derivedgrowth factor-A (PDGF-A), PDGF-B, PDGF-C, PDGF-D, macrophage migrationinhibitory factor (MIF), IGF-1, hepatocyte growth factor (HGF), PDGF-Rα,PDGF-Rβ, CXCR4, C-C chemokine receptor type 1 (CCR1), IGF-1 receptor(IGF-1R), hepatocyte growth factor receptor (HGFR), CXCL12 and NFkappaBand do not express detectable levels of CD14, CD34 and CD45.

If the damaged tissue is retinal tissue, the PMLs in the populationpreferably express detectable levels of CD29, CD44, CD73, CD90, CD105,CD271, CXCR4, vascular endothelial growth factor (VEGF), transforminggrowth factor beta 1 (TGF-beta 1), insulin-like growth factor-1 (IGF-1),fibroblast growth factor (FGF), tumour necrosis factor alpha(TNF-alpha), interferon gamma (IFN-gamma), interleukin-1 alpha (IL-1alpha), CXCL12, CD109, CD119, nuclear factor kappa-light-chain-enhancerof activated B cells (NFkappa B), CD140a, CD140b, CD221, CD222, CD304,CD309 and CD325 and do not express detectable levels of CD14, CD34 andCD45.

In all instances, the PMLs of the invention are preferably derived fromthe patient or an allogeneic donor. Deriving the PMLs of the inventionfrom the patient should ensure that the PMLs are themselves not rejectedby the patient's immune system. Any difference between the donor andrecipient will ultimately cause clearance of the PMLs, but not beforethey have repaired at least a part of the damaged tissue.

The invention concerns administering to the patient a therapeuticallyeffective number of PMLs of the invention to the patient. Atherapeutically effective number is a number which ameliorates one ormore symptoms of the damage, disease or injury. A therapeuticallyeffective number is preferably a number which repairs the damaged tissueor treats the disease or injury. Suitable numbers are discussed in moredetail below.

The PMLs of the invention may be administered to any suitable patient.The patient is generally a human patient. The patient may be an infant,a juvenile or an adult. The patient may be known to have a damagedtissue or is suspected of having a damaged tissue. The patient may besusceptible to, or at risk from, the relevant disease or injury. Forinstance, the patient may be genetically predisposed to heart failure.

The invention may be used in combination with other means of, andsubstances for, repairing damaged tissue or providing pain relief. Insome cases, the PMLs of the invention may be administeredsimultaneously, sequentially or separately with other substances whichare intended for repairing the damaged tissue or for providing painrelief. The PMLs may be used in combination with existing treatments fordamaged tissue and may, for example, be simply mixed with suchtreatments. Thus the invention may be used to increase the efficacy ofexisting treatments of damaged tissue.

The invention preferably concerns the use of PMLs loaded or transfectedwith a therapeutic and/or diagnostic agent. A therapeutic agent may helpto repair the damaged tissue. A diagnostic agent, such as a fluorescentmolecule, may help to identify the location of the PMLs in the patient.The PMLs may be loaded or transfected using any method known in the art.The loading of PMLs may be performed in vitro or ex vivo. In each case,the PMLs may simply be in contact with the agent in culture.Alternatively, the PMLs may be loaded with an agent using deliveryvehicle, such as liposomes. Such vehicles are known in the art.

The transfection of PMLs may be performed in vitro or ex vivo.Alternatively, stable transfection may be performed at the MC stageallowing PMLs expressing the transgene to be differentiated from them.The PMLs are transfected with a nucleic acid encoding the agent. Forinstance, viral particles or other vectors encoding the agent may beemployed. Methods for doing this are known in the art.

The nucleic acid gives rise to expression of the agent in the PMLs. Thenucleic acid molecule will preferably comprise a promoter which isoperably linked to the sequences encoding the agent and which is activein the PMLs or which can be induced in the PMLs.

In a particularly preferred embodiment, the nucleic acid encoding theagent may be delivered via a viral particle. The viral particle maycomprise a targeting molecule to ensure efficient transfection. Thetargeting molecule will typically be provided wholly or partly on thesurface of the virus in order for the molecule to be able to target thevirus to the PMLs.

Any suitable virus may be used in such embodiments. The virus may, forexample, be a retrovirus, a lentivirus, an adenovirus, anadeno-associated virus, a vaccinia virus or a herpes simplex virus. In aparticularly preferred embodiment the virus may be a lentivirus. Thelentivirus may be a modified HIV virus suitable for use in deliveringgenes. The lentivirus may be a SIV, FIV, or equine infectious anemiavirus (EQIA) based vector. The virus may be a moloney murine leukaemiavirus (MMLV). The viruses used in the invention are preferablyreplication deficient.

Viral particles do not have to be used. Any vector capable oftransfecting the PMLs of the invention may be used, such as conventionalplasmid DNA or RNA transfection.

Uptake of nucleic acid constructs may be enhanced by several knowntransfection techniques, for example those including the use oftransfection agents. Examples of these agents includes cationic agents,for example, calcium phosphate and DEAE-Dextran and lipofectants, forexample, lipofectAmine, fugene and transfectam.

The cell may be loaded or transfected under suitable conditions. Thecell and agent or vector may, for example, be contacted for between fiveminutes and ten days, preferably from an hour to five days, morepreferably from five hours to two days and even more preferably fromtwelve hours to one day.

The invention also provides PMLs which have been loaded or transfectedwith an agent as discussed above. Such PMLs may be used in thetherapeutic embodiments of the invention.

In some embodiments, MCs may be recovered from a patient, converted intoPMLs using the invention, loaded or transfected in vitro and thenreturned to the same patient. In such instances, the PMLs employed inthe invention, will be autologous cells and fully matched with thepatient. In a preferred case, the cells employed in the invention arerecovered from a patient and utilised ex vivo and subsequently returnedto the same patient.

Pharmaceutical Compositions and Administration

The invention additionally provides a pharmaceutical compositioncomprising (a) a PML of the invention or a population of the inventionand (b) a pharmaceutically acceptable carrier or diluent. Thecomposition may comprise any of the PMLs or populations mentioned hereinand, in some embodiments, the nucleic acid molecules, vectors, orviruses described herein. The invention provides a method of repairing adamaged tissue in a patient comprising administering to the patient aneffective amount of a pharmaceutical composition of the invention. Anyof the therapeutic embodiments discussed above equally apply to thisembodiment.

The various compositions of the invention may be formulated using anysuitable method. Formulation of cells with standard pharmaceuticallyacceptable carriers and/or excipients may be carried out using routinemethods in the pharmaceutical art. The exact nature of a formulationwill depend upon several factors including the cells to be administeredand the desired route of administration. Suitable types of formulationare fully described in Remington's Pharmaceutical Sciences, 19^(th)Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

The cells may be administered by any route. Suitable routes include, butare not limited to, intravenous, intramuscular, intraperitoneal or otherappropriate administration routes. If the damaged tissue is retinaltissue, the cells may be administered to the eye. If the damaged tissueis cardiac tissue, the cells may be administered via an endomyocardial,epimyocardial, intraventicular, intracoronary, retrograde coronarysinus, intra-arterial, intra-pericardial or intravenous route. If thedamaged tissue is bone, the cells may be administered via anintraosseous route or to the site of the injury, such as a fracture, ordisease. The cells are preferably administered intravenously.

Compositions may be prepared together with a physiologically acceptablecarrier or diluent. Typically, such compositions are prepared as liquidsuspensions of cells. The cells may be mixed with an excipient which ispharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,of the like and combinations thereof.

In addition, if desired, the pharmaceutical compositions of theinvention may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, and/or adjuvantswhich enhance effectiveness. The composition preferably comprises humanserum albumin.

One suitable carrier or diluents is Plasma-Lyte A®. This is a sterile,nonpyrogenic isotonic solution for intravenous administration. Each 100mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of SodiumGluconate (C6H11NaO7); 368 mg of Sodium Acetate Trihydrate, USP(C2H3NaO2.3H2O); 37 mg of Potassium Chloride, USP (KCl); and 30 mg ofMagnesium Chloride, USP (MgCl2.6H2O). It contains no antimicrobialagents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to8.0).

The PMLs are administered in a manner compatible with the dosageformulation and in such amount will be therapeutically effective. Thequantity to be administered depends on the subject to be treated,capacity of the subject's immune system and the degree repair desired.Precise amounts of PMLs required to be administered may depend on thejudgement of the practitioner and may be peculiar to each subject.

Any suitable number of cells may be administered to a subject. Forexample, at least, or about, 0.5×10⁶, 1.5×10⁶, 4.0×10⁶ or 5.0×10⁶ cellsper kg of patient may administered. For example, at least, or about,10⁵, 10⁶, 10⁷, 10⁸, 10⁹ cells may be administered. As a guide, thenumber of cells of the invention to be administered may be from 10⁵ to10⁹, preferably from 10⁶ to 10⁸. Typically, up to 2×10⁸ PMLs areadministered to each patient. Any of the specific numbers discussedabove with reference to the populations of the invention may beadministered. In such cases where cells are administered or present,culture medium may be present to facilitate the survival of the cells.In some cases the cells of the invention may be provided in frozenaliquots and substances such as DMSO may be present to facilitatesurvival during freezing. Such frozen cells will typically be thawed andthen placed in a buffer or medium either for maintenance or foradministration.

The following Example illustrates the invention.

Example Materials and Methods

Once blood was taken from patients, progenitor cells of mesodermalorigin were prepared in a stem cell laboratory under hygienicconditions; open containers with cells or other material were handledunder a laminar-flow hood. At each preparation step, samples were drawnand stem cell number and viability was determined.

Mononuclear cells were isolated from whole blood by Ficoll-Paque® 1.073density centrifugation and were cultured in α-MEM-PL for 5 days.Adherent cells were harvested and their immunophenotypes were determinedby immunofluorescence staining for a number of cellular markers (seebelow) by flow cytometric analysis. Appropriate isotype controls wereused for each staining procedure.

Cell viability was assessed with 1% trypan blue solution. Cells will beenumerated by FACS (FACSCalibur, Beckton Dickinson). Cells were alsotested for mycoplasma, sterility (assessed by gram stain), endotoxin,identity, purity, and viability and karyotyping to exclude chromosomalabnormalities.

The following summarises how the cells were actually derived.

1. 20 ml of peripheral blood was taken from the patient.2. 11.5 ml of remaining blood was then passed through the Ficoll Paque®1.073.3. This was centrifuged to give mononuclear cells these were eitherthen:4a. Grown in culture for 8 days in 0% oxygen or4b. Run through Rosette Separation and then grown in culture for 8 daysin 0% oxygen.5. Media was changed and cells were in culture for 14 days in 0% oxygen.6. The cells were then harvested and then run through FACS.A variety of markers were investigated using RT-PCR and FACS analysis.The main markers investigated were CD14, CD29, CD34, CD44, CD45, CD73,CD90, CD105, CD271, CD181, CD182 and CD184.The following is the working protocol that was used.

Platelet-Rich Plasma (PRP) Preparation

1. The blood sample was divided into two 15 ml Falcon tubes, >8 ml ineach.

2. Centrifuged at 120×g, 15 min, no brake, room temperature (RT).

3. The platelet-rich plasma (PRP) supernatant was transferred to a new15 ml Falcon tube.

4. The volume of the transferred PRP was noted.

5. The volume of PRP was replaced with Hank's Balanced Salt Solution(HBSS).

Culture Media Preparation

-   -   1. 0.3 ml PRP was transferred to an eppendorf tube for automatic        haematology analysis using the Cell-Dyn instrument. The        theoretical maximum number of platelets was calculated.        Theoretical maximum number of platelets=Platelet        concentration×PRP volume    -   2. 0.25 ml PRP was transferred to an eppendorf tube for        cryopreservation (−80° C.).    -   3. The remaining PRP was centrifuged at 1610×g, 10 min, RT, with        brake.    -   4. The platelet free plasma (PFP) supernatant was removed into a        separate falcon tube and the platelet pellet was re-suspended        with a volume of PFP that gives a concentration of 1×10⁹        cells/ml (using the theoretical max number of platelets—aim for        1.5×10⁹ to achieve 1×10⁹).    -   5. The remaining PFP was transferred into eppendorf tubes in        0.25 ml aliquots for cryopreservation (−80° C.).    -   6. The lid of the PRP falcon tube was wrapped in parafilm.    -   7. The falcon tube was submerged in liquid nitrogen for 5 mins.    -   8. The falcon tube was submerged in 37° C. water bath until        thawed.    -   9. Steps 7 and 8 were repeated a further 3 times.    -   10. Culture media was made up by adding the PL at 10% to αMEM,        5U/ml Heparin, 2 mM glutamax, 1% P/S (i.e. add 1.5 ml PL to 13.5        ml media).

MNC Isolation

-   -   1. The diluted blood sample volume (16.5 ml) was combined into a        new 50 ml Falcon tube.    -   2. The blood sample was further diluted 1:2 with HBSS to −33 ml.    -   3. 15 ml of Ficoll-Paque PREMIUM 1.073 was added to two new 50        ml Falcon tubes.    -   4. The diluted blood sample was carefully layered on top of the        Ficoll-Paque by tilting the tube and ejecting the sample slowly        against the tube wall.    -   5. This was centrifuged at 400×g, 35 min, no brake, RT.    -   6. As much of the supernatant (the HBSS) as possible was        disgarded without interrupting the cloudy mononuclear cell layer        with the help of a soft Pasteur pipette.    -   7. The cloudy mononuclear cell layer that is resting on top of        the clear Ficoll-Paque was aspirated to a new 50 ml tube,        pooled!    -   8. The volume of transferred MNCs was noted by aspirating it        into a 10 ml pipette.    -   9. Half of the volume was transferred to a new 50 ml tube.    -   10. The MNCs were diluted in both tubes with at least 3× the        sample volume with HBSS, about 13 ml.    -   11. Both tubes were centrifuged at 500×g, 15 min, with brake,        RT.    -   12. The supernatant was discarded.    -   13. One of the tubes was resuspended to approximately 1 million        MNCs/ml, about 5 ml.

Rosette-Sep Enrichment

-   -   1. The second MNC pellet was resuspended with 660 μL whole blood        from the initial 0.75 ml aliquot.    -   2. 33 μL Rosette-Sep was added and mixed by pipetting.    -   3. This was incubated for 20 minutes, room temperature.    -   4. The sample was diluted 1:2 by adding 700 μL HBSS, to a total        sample volume of ˜1.4 ml.    -   5. 1 ml of Ficoll-Paque was added to a new 15 ml Falcon tube.    -   6. The diluted blood sample was carefully layered on top of the        Ficoll-Paque.    -   7. This was centrifuged at 400×g, 35 min, no brake, RT.    -   8. The supernatant was discarded by aspirating it with a 1 ml        single-channel pipette.    -   9. The cloudy cell layer on top of the Ficoll was transferred to        a new 15 ml Falcon tube.    -   10. The volume of enriched cells was noted and they were diluted        with at least 3× the sample volume with HBSS, about 3 ml.    -   11. The cells were centrifuged at 500×g, 15 min, with brake, RT.    -   12. The supernatant was discarded.    -   13. The pellet was resuspended in 0.5 ml.

Cell Culture

-   -   1. The cells were seeded at a seeding density of 1.0×10⁵ cells        into either autologous or allogeneic platelet lysate medium.    -   2. This was topped up with a suitable medium volume.    -   3. The cells were incubated in 37° C., 0% 02, 5% CO₂ for 8 days.    -   4. The media were changed on day 8 and the cells were incubated        until day 14.    -   5. The colonies were picked and transferred to new culture        vessels. Allogeneic platelet lysate medium were added.    -   6. The culturing and passaging of the cells was continued until        approximately 5×10⁵-1×10⁶ cells were obtained.    -   7. The cells were harvested and analysed by flow cytometry and,        as necessary, the cells were cryopreserved.

Homing and Anti-Inflammatory Tests

The cells produced in accordance with the invention were tested fortheir ability to home to specific, damaged tissues in mice and induceanti-inflammatory effects once there. For homing, cells were labelledwith fluorescent agent and their location in the mouse body determinedusing bioluminescence.

For anti-inflammatory effects, enzyme-linked immunosorbent assays(ELISAs) for various inflammatory markers, including interleukins (suchas IL-8), selectins, adhesion molecules (such as ICAM-1) andchemoattractant proteins (such as MCP-1 and TNF-α), were performed.

Results All Cells

All progenitor cells of mesodermal lineage produced in accordance withthe invention expressed CD29, CD44, CD73, CD90, CD105 and CD271, but didnot express CD14, CD34 and CD45. An exemplary RT-PCR gel showing thepresence of CD44 and the absence of CD34 is shown in FIG. 1.

Exemplary sets of FACS results are shown in FIGS. 2 to 4. These confirmsthat the cells are cells are positive for at least CD73 and CD90 andnegative for CD14, CD34 and CD45.

The cells are typically from 10 to 20 μm in diameter. The cellstypically have a spindle-shaped morphology and are fibroblast like (i.e.they have a small cell body with a few cell processes that are long andthin).

Homing Cells

Cells capable of homing to specific damaged tissues were shown toexpress chemokine receptor types 1 and 2 (CXCR1 and CXCR2).

Cells capable of homing to damaged heart tissue and bone tissues wereshown to express CXCR4.

The above table shows BLI signal semiquantitative analysis. Signal atthe fracture tibia site region of interest (ROI), measured asphotons/seconds/cm2/sr, was normalized by subtracting the backgroundsignal found in an equal ROI in the contra-lateral unfractured tibia. a,p<0.05 versus CXCR4 group; b, p<0.01 versus CXCR4 group; c, p<0.05versus PMLs by Tukey post-test. Abbreviations: ANOVA, analysis ofvariance; PML, progenitor cell of mesodermal lineage.

Cells capable of homing to damaged retinal tissue were shown to expressCXCR4, vascular endothelial growth factor (VEGF), transforming growthfactor beta 1 (TGF-beta 1), insulin-like growth factor-1 (IGF-1),fibroblast growth factor (FGF), tumour necrosis factor alpha(TNF-alpha), interferon gamma (IFN-gamma), interleukin-1 alpha (IL-1alpha), CXCL12, CD109, CD119, nuclear factor kappa-light-chain-enhancerof activated B cells (NFkappa B), CD140a, CD140b, CD221, CD222, CD304,CD309 and CD325.

Cells capable of homing to damaged bone tissue and the cell expressesdetectable levels of TGF-beta 3, bone morphogenetic protein-6 (BMP-6),SOX-9, Collagen-2, CD117 (c-kit), chemokine (C-C motif) ligand 12(CCL12), CCL7, interleukin-8 (IL-8), platelet-derived growth factor-A(PDGF-A), PDGF-B, PDGF-C, PDGF-D, macrophage migration inhibitory factor(MIF), IGF-1, hepatocyte growth factor (HGF), PDGF-Rα, PDGF-Rβ, CXCR4,C-C chemokine receptor type 1 (CCR1), IGF-1 receptor (IGF-1R),hepatocyte growth factor receptor (HGFR), CXCL12 and NFkappaB.

Anti-Inflammatory Effects

The cells produced in accordance with the invention were shown toexpress the following anti-inflammatory markers: CD120a (tumour-necrosisfactor (TNF)-alpha Receptor 1), CD120b (TNF-alpha Receptor 2), CD50(Intercellular Adhesion Molecule-3, ICAM-3), CD54 (ICAM-1), CD58(Lymphocyte function-associated antigen-1, LFA-1), CD62E (E-selectin),CD62L (L-selectin), CD62P (P-selectin), CD106 (Vascular cell adhesionprotein, VCAM-1), CD102 (ICAM-2), CD166 (Activated leukocyte celladhesion molecule), CD104 (Beta 4 integrin), CD123 (Interleukin-3Receptor), CD124 (Interleukin-4 Receptor), CD126 (Interleukin-6Receptor), CD127 (Interleukin-7 Receptor) and fibroblast growth factorreceptor (FGFR).

1. A progenitor cell of mesodermal lineage, wherein the cell (a)expresses detectable levels of CD29, CD44, CD73, CD90, CD105 and CD271and (b) does not express detectable levels of CD14, CD34 and CD45.
 2. Aprogenitor cell according to claim 1, wherein the cell is capable ofmigrating to a specific, damaged tissue in a patient.
 3. A progenitorcell according to claim 2, wherein the cell expresses detectable levelsof C-X-C chemokine receptor type 1 (CXCR1).
 4. A progenitor cellaccording to claim 2 or 3, wherein the cell expresses detectable levelsof C-X-C chemokine receptor type 2 (CXCR2).
 5. A progenitor cellaccording to any one of claims 2 to 4, wherein the specific tissue iscardiac tissue, retinal tissue or bone tissue.
 6. A progenitor cellaccording to claim 5, wherein the specific tissue is heart tissue orbone tissue and the cell expresses detectable levels of C-X-C chemokinereceptor type 4 (CXCR4).
 7. A progenitor cell according to claim 5,wherein the specific tissue is retinal tissue and the cell expressesdetectable levels of CXCR4, vascular endothelial growth factor (VEGF),transforming growth factor beta 1 (TGF-beta 1), insulin-like growthfactor-1 (IGF-1), fibroblast growth factor (FGF), tumour necrosis factoralpha (TNF-alpha), interferon gamma (IFN-gamma), interleukin-1 alpha(IL-1 alpha), CXCL12, CD109, CD119, nuclear factorkappa-light-chain-enhancer of activated B cells (NFkappa B), CD140a,CD140b, CD221, CD222, CD304, CD309 and CD325.
 8. A progenitor cellaccording to claim 5, wherein the specific tissue is bone tissue and thecell expresses detectable levels of TGF-beta 3, bone morphogeneticprotein-6 (BMP-6), SOX-9, Collagen-2, CD117 (c-kit), chemokine (C-Cmotif) ligand 12 (CCL12), CCL7, interleukin-8 (IL-8), platelet-derivedgrowth factor-A (PDGF-A), PDGF-B, PDGF-C, PDGF-D, macrophage migrationinhibitory factor (MIF), IGF-1, hepatocyte growth factor (HGF), PDGF-Rα,PDGF-Rβ, CXCR4, C-C chemokine receptor type 1 (CCR1), IGF-1 receptor(IGF-1R), hepatocyte growth factor receptor (HGFR), CXCL12 and NFkappaB.9. A progenitor cell according to any one of the preceding claims,wherein the cell is capable of having anti-inflammatory effects in adamaged tissue in a patient.
 10. A progenitor cell according to claim 9,wherein the cell expresses detectable levels of CD120a (tumour-necrosisfactor (TNF)-alpha Receptor 1), CD120b (TNF-alpha Receptor 2), CD50(Intercellular Adhesion Molecule-3, ICAM-3), CD54 (ICAM-1), CD58(Lymphocyte function-associated antigen-1, LFA-1), CD62E (E-selectin),CD62L (L-selectin), CD62P (P-selectin), CD106 (Vascular cell adhesionprotein, VCAM-1), CD102 (ICAM-2), CD166 (Activated leukocyte celladhesion molecule), CD104 (Beta 4 integrin), CD123 (Interleukin-3Receptor), CD124 (Interleukin-4 Receptor), CD126 (Interleukin-6Receptor), CD127 (Interleukin-7 Receptor) and fibroblast growth factorreceptor (FGFR).
 11. A progenitor cell according to any one of thepreceding claims, wherein the cell expresses detectable levels of one ormore of (i) insulin-like growth factor-1 (IGF-1), (ii) IGF-1 receptor;(iii) C-C chemokine receptor type 1 (CCR1), (iv) stromal cell-derivedfactor-1 (SDF-1), (v) hypoxia-inducible factor-1 alpha (HIF-1 alpha),(vi) Akt1 and (vii) hepatocyte growth factor (HGF) and/or granulocytecolony-stimulating factor (G-CSF).
 12. A progenitor cell according toclaim 11, wherein the cell overexpresses one or more of (i) to (vii).13. A progenitor cell according to any one of the preceding claims,wherein the cell expresses detectable levels of one or more of (i)vascular endothelial growth factor (VEGF), (ii) transforming growthfactor beta (TGF-beta), (iii) insulin-like growth factor-1 (IGF-1), (iv)fibroblast growth factor (FGF), (v) tumour necrosis factor alpha(TNF-alpha), (vi) interferon gamma (IFN-gamma) and (vii) interleukin-1alpha (IL-1 alpha)
 14. A progenitor cell according to claim 13, whereinthe cell overexpresses one or more of (i) to (vii)
 15. A progenitor cellaccording to any one of the preceding claims, wherein the cell isautologous.
 16. A progenitor cell according to any one of claims 1 to14, wherein the cell is allogeneic.
 17. A population comprising two ormore progenitor cells of mesodermal lineage as defined in any one ofclaims 1 to
 16. 18. A population according to claim 15, wherein thepopulation comprises at least about 5×10⁵ cells as defined in any one ofclaims 1 to
 16. 19. A pharmaceutical composition comprising (a) aprogenitor cell according to any one of claims 1 to 16 or a populationaccording to claim 17 or 18 and (b) a pharmaceutically acceptablecarrier or diluent.
 20. A method of producing a population of progenitorcells of mesodermal lineage according to claim 17 or 18, comprising (a)culturing mononuclear cells (MCs) under conditions which induce the MCsto differentiate into progenitor cells of mesodermal lineage and (b)harvesting and culturing those progenitor cells which have an expressionpattern as defined in any one of claims 1, 3, 4, 6, 7, 8 and 10 to 14and thereby producing a population according to claim 17 or
 18. 21. Amethod according to claim 20, wherein the MCs are peripheral bloodmononuclear cells (PBMCs).
 22. A method according to claim 20 or 21,wherein step (a) comprises culturing the MCs under conditions that allowthe progenitor cells to adhere.
 23. A method according to any one ofclaims 20 to 22, wherein steps (a) and/or (b) comprise culturing theMSCs and/or progenitor cells with platelet lysate.
 24. A methodaccording to any one of claims 20 to 23, wherein steps (a) and/or (b)comprise culturing the MSCs and/or progenitor cells under low oxygenconditions.
 25. A method according to claim 24, wherein the low oxygenconditions are less about than 20% oxygen (02).
 26. A method accordingto any one of claims 20 to 25, wherein the MCs are obtained from apatient or an allogeneic donor.
 27. A method of repairing a damagedtissue in a patient, comprising administering to the patient apopulation according to claim 17 or 18, wherein the population comprisesa therapeutically effective number of cells, and thereby treating thedamaged tissue in the patient.
 28. A method according to claim 27,wherein the tissue is derived from the mesoderm.
 29. A method accordingto claim 28, wherein the tissue is cardiac tissue, retinal tissue orbone tissue.
 30. A method according to claim 29, wherein the method is(a) for repairing damaged cardiac tissue and the population comprises atherapeutically effective amount of cells as defined in claim 6, (b) forrepairing damaged retinal tissue and the population comprises atherapeutically effective amount of cells as defined in claim 7 or (c)for repairing damaged bone tissue and the population comprises atherapeutically effective amount of cells as defined in claim
 8. 31. Amethod according to any one of claims 27 to 30, wherein the tissue isdamaged by injury or disease.
 32. A method according to claim 27,wherein the method is for treating a cardiac injury or disease,age-related macular degeneration or a bone injury or disease in thepatient.
 33. A method according to claim 32, wherein the cardiac injuryor disease is selected from myocardial infarct, left ventricularhypertrophy, right ventricular hypertrophy, emboli, heart failure,congenital heart deficit, heart valve disease, arrhythmia andmyocarditis.
 34. A method according to claim 32, wherein the bone injuryor disease is selected from fracture, Salter-Harris fracture, greenstickfracture, bone spur, craniosynostosis, Coffin-Lowry syndrome,fibrodysplasia ossificans progressive, fibrous dysplasia, Fong Disease(or Nail-patella syndrome), hypophosphatasia, Klippel-Feil syndrome,Metabolic Bone Disease, Nail-patella syndrome, osteoarthritis, osteitisdeformans (or Paget's disease of bone), osteitis fibrosa cystica (orOsteitis fibrosa or Von Recklinghausen's disease of bone), osteitispubis, condensing osteitis (or osteitis condensans), osteitis condensansilii, osteochondritis dissecans, osteogenesis imperfecta, osteomalacia,osteomyelitis, osteopenia, osteopetrosis, osteoporosis, osteonecrosis,porotic hyperostosis, primary hyperparathyroidism, renal osteodystrophy,bone cancer, a bone lesion associated with metastatic cancer, GorhamStout disease, primary hyperparathyroidism, periodontal disease, andaseptic loosening of joint replacements.
 35. A method according to anyone of claims 32 to 34, wherein the method is (a) for treating a cardiacinjury or disease and the population comprises a therapeuticallyeffective amount of cells as defined in claim 6, (b) for treatingage-related macular degeneration and the population comprises atherapeutically effective amount of cells as defined in claim 7 or (c)for treating a bone injury or disease and the population comprises atherapeutically effective amount of cells as defined in claim
 8. 36. Amethod according to any one of claims 27 to 35, wherein the populationis produced using MCs obtained from the patient or an allogeneic donor.37. A population according to claim 17 or 18 for use in repairing adamaged tissue in a patient.
 38. A population according to claim 17 or18 for use in treating a cardiac injury or disease, age-related maculardegeneration or a bone injury or disease in a patient in need thereof.